Monoclonal antibodies and methods for their use in the detection of cervical disease

ABSTRACT

Compositions and methods for diagnosing high-grade cervical disease in a patient sample are provided. The compositions include novel monoclonal antibodies, and variants and fragments thereof, that specifically bind to MCM2. Monoclonal antibodies having the binding characteristics of an MCM2 antibody of the invention are further provided. Hybridoma cell lines that produce an MCM2 monoclonal antibody of the invention are also disclosed herein. The compositions find use in practicing methods for diagnosing high-grade cervical disease comprising detecting overexpression of MCM2 in a cervical sample from a patient. Kits for practicing the methods of the invention are further provided. Polypeptides comprising the amino acid sequence for an MCM2 epitope and methods of using these polypeptides in the production of antibodies are also encompassed by the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/675,305, filed Apr. 27, 2005, and U.S. Provisional ApplicationSer. No. 60/718,082, filed Sep. 16, 2005, both of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to antibodies capable of binding to MCM2 andmethods of using these antibodies, particularly in the diagnosis ofcervical disease.

BACKGROUND OF THE INVENTION

Carcinoma of the cervix is the second most common neoplasm in women,accounting for approximately 12% of all female cancers and causingapproximately 250,000 deaths per year. Baldwin et al. (2003) NatureReviews Cancer 3:1-10. In many developing countries where mass screeningprograms are not available, the clinical problem is more serious.Cervical cancer in these countries is the number one cause of cancerdeaths in women.

The majority of cases of cervical cancer represent squamous cellcarcinoma, although adenocarcinoma is also seen. Cervical cancer can beprevented by population screening as it evolves through well-definednoninvasive intraepithelial stages, which can be distinguishedmorphologically. Williams et al. (1998) Proc. Natl. Acad. Sci. USA95:14932-14937. While it is not understood how normal cells becometransformed, the concept of a continuous spectrum of histopathologicalchange from normal, stratified epithelium through cervicalintraepithelial neoplasia (CIN) to invasive cancer has been widelyaccepted for years. The precursor to cervical cancer is dysplasia, alsoknown in the art as CIN or squamous intraepithelial lesions (SIL).Squamous intraepithelial abnormalities may be classified by using thethree-tiered (CIN) or two-tiered (Bethesda) system. Under the Bethesdasystem, low-grade squamous intraepithelial lesions (LSIL), correspondingto CINI and HPV infection, generally represent productive HPV infectionswith a relatively low risk of progression to invasive disease.High-grade squamous intraepithelial lesions (HSIL), corresponding toCINII and CINIII in the three-tiered system, show a higher risk ofprogression to cervical cancer than do LSIL, although both LSIL and HSILare viewed as potential precursors of malignancy. Patient samples mayalso be classified as ASCUS (atypical squamous cells of unknownsignificance) or AGUS (atypical glandular cells of unknown significance)under this system.

A strong association of cervical cancer and infection by high-risk typesof human papilloma virus (HPV), such as types 16, 18, and 31, has beenestablished. In fact, a large body of epidemiological and molecularbiological evidence has established HPV infection as a causative factorin cervical cancer. Moreover, HPV is found in 85% or more of the casesof high-grade cervical disease. However, HPV infection is very common,possibly occurring in 5-15% of women over the age of 30, but fewHPV-positive women will ever develop high-grade cervical disease orcancer. The presence of HPV alone is indicative only of infection, notof high-grade cervical disease, and, therefore, testing for HPVinfection alone results in many false positives. See, for example,Wright et al. (2004) Obstet. Gynecol. 103:304-309.

Current literature suggests that HPV infects the basal stem cells withinthe underlying tissue of the uterine-cervix. Differentiation of the stemcells into mature keratinocytes, with resulting migration of the cellsto the stratified cervical epithelium, is associated with HPV viralreplication and re-infection of cells. During this viral replicationprocess, a number of cellular changes occur that include cell-cyclede-regulation, active proliferation, DNA replication, transcriptionalactivation and genomic instability (Crum (2000) Modern Pathology13:243-251; Middleton et al. (2003) J. Virol. 77:10186-10201; Pett etal. (2004) Cancer Res. 64:1359-1368).

Most HPV infections are transient in nature, with the viral infectionresolving itself within a 12-month period. For those individuals whodevelop persistent infections with one or more oncogenic subtypes ofHPV, there is a risk for the development of neoplasia in comparison topatients without an HPV infection. Given the importance of HPV in thedevelopment of cervical neoplasia, the clinical detection of HPV hasbecome an important diagnostic tool in the identification of patients atrisk for cervical neoplasia development. The clinical utility ofHPV-based screening for cervical disease is in its negative predictivevalue. An HPV negative result in combination with a history of normalPap smears is an excellent indicator of a disease-free condition and alow risk of cervical neoplasia development during the subsequent 1-3years. However, a positive HPV result is not diagnostic of cervicaldisease; rather it is an indication of infection. Although the majorityof HPV infections is transient and will spontaneously clear within a12-month period, a persistent infection with a high-risk HPV viralsubtype indicates a higher risk for the development of cervicalneoplasia. To supplement HPV testing, the identification of molecularmarkers associated with cervical neoplasia is expected to improve theclinical specificity for cervical disease diagnosis.

Cytological examination of Papanicolaou-stained cervical smears (Papsmears) currently is the method of choice for detecting cervical cancer.The Pap test is a subjective method that has remained substantiallyunchanged for 60 years. There are several concerns, however, regardingits performance. The reported sensitivity of a single Pap test (theproportion of disease positives that are test-positive) is low and showswide variation (30-87%). The specificity of a single Pap test (theproportion of disease negatives that are test-negative) might be as lowas 86% in a screening population and considerably lower in the ASCUSPLUS population for the determination of underlying high-grade disease.See, Baldwin et al., supra. A significant percentage of Pap smearscharacterized as LSIL or CINI are actually positive for high-gradelesions. Furthermore, up to 10% of Pap smears are classified as ASCUS(atypical squamous cells of undetermined significance), i.e., it is notpossible to make a clear categorization as normal, moderate or severelesion, or tumor. However, experience shows that up to 10% of this ASCUSpopulation has high-grade lesions, which are consequently overlooked.See, for example, Manos et al. (1999) JAMA 281:1605-1610. Therefore,molecular biomarkers that are selectively overexpressed in high-gradecervical disease and compositions for the detection of these biomarkersare needed to practice reliable methods for diagnosing high-gradecervical disease.

Minichromosome maintenance (MCM) proteins play an essential part ineukaryotic DNA replication. The minichromosome maintenance (MCM)proteins function in the early stages of DNA replication through loadingof the prereplication complex onto DNA and functioning as a helicase tohelp unwind the duplex DNA during de novo synthesis of the duplicate DNAstrand. Each of the MCM proteins has DNA-dependent ATPase motifs intheir highly conserved central domain. Levels of MCM proteins generallyincrease in a variable manner as normal cells progress from G0 into theG1/S phase of the cell cycle. In the G0 phase, MCM2 and MCM5 proteinsare much less abundant than are the MCM7 and MCM3 proteins. MCM6 forms acomplex with MCM2, MCM4, and MCM7, which binds histone H3. In addition,the subcomplex of MCM4, MCM6, and MCM7 has helicase activity, which ismediated by the ATP-binding activity of MCM6 and the DNA-bindingactivity of MCM4. See, for example, Freeman et al. (1999) Clin. CancerRes. 5:2121-2132; Lei et al. (2001) J. Cell Sci. 114:1447-1454; Ishimiet al. (2003) Eur. J. Biochem. 270:1089-1101, all of which are hereinincorporated by reference in their entirety.

Early publications have shown that the MCM proteins, and in particular,MCM-5, are useful for the detection of cervical disease (Williams et al.(1998) Proc Natl Acad Sci U.S.A. 95:14932-14937), as well as othercancers (Freeman et al. (1999) Clin Cancer Res. 5:2121-2132). Thepublished literature indicates that antibodies to MCM-5 are capable ofdetecting cervical neoplastic cells. The specificity for detection ofhigh-grade cervical disease has not been demonstrated for MCM-5(Williams et al. (1998) Proc Natl Acad Sci U.S.A. 95:14932-14937). Thedetection of MCM-5 expression is not restricted to high-grade cervicaldisease but is also detected in identified low-grade dysplasia andproliferative cells that have re-entered the cell cycle followinginfection with high-risk HPV. In addition to MCM-5, other members fromthe MCM family, including MCM-2 and MCM-7 have been shown to bepotentially useful markers for the detection of cervical neoplasia intissue samples (Freeman et al. (1999) Clin Cancer Res. 5:2121-2132;Brake et al. (2003) Cancer Res. 63:8173-8180). Recent results have shownthat MCM-7 appears to be a specific marker for the detection ofhigh-grade cervical disease using immunochemistry formats (Brake et al.(2003) Cancer Res. 63:8173-8180; Malinowski et al. (2004) Acta Cytol.43:696).

Therefore, there is a need in the art for antibodies that are capable ofdetecting expression of a biomarker that is selectively overexpressed inhigh-grade cervical disease. Such antibodies could be used in methodsfor differentiating high-grade disease from conditions that are notconsidered clinical disease, such as early-stage HPV infection and milddysplasia.

SUMMARY OF THE INVENTION

Compositions and methods for diagnosing high-grade cervical disease areprovided. Compositions include monoclonal antibodies capable of bindingto nuclear biomarker proteins of the invention, particularly MCMproteins, more particularly MCM2. Antigen-binding fragments and variantsof these monoclonal antibodies, hybridoma cell lines capable ofproducing these antibodies, and kits comprising the monoclonalantibodies of the invention are also encompassed herein.

The compositions of the invention find use in methods for diagnosinghigh-grade cervical disease. The methods comprise detectingoverexpression of at least one nuclear biomarker, wherein overexpressionof the nuclear biomarker is indicative of high-grade cervical disease.Specifically, the methods comprise using the antibodies of the inventionto detect overexpression of MCM2 in a cervical sample.

Compositions of the invention further include isolated polypeptides thatcomprise an epitope capable of binding an MCM2 monoclonal antibody.These polypeptides find use in methods for producing MCM2 antibodies.Isolated nucleic acid molecules encoding the amino acid sequences of theMCM2 epitopes are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for diagnosing high-grade cervical disease areprovided. Compositions include monoclonal antibodies that are capable ofbinding to nuclear biomarker proteins that are selectively overexpressedin high-grade cervical disease, particularly MCM proteins, moreparticularly MCM2. Hybridoma cell lines that produce the monoclonalantibodies of the present invention are also disclosed. Kits comprisingthe monoclonal antibodies described herein are further provided. Thepresent compositions find use in methods for diagnosing high-gradecervical disease in a patient.

The compositions of the invention include monoclonal antibodies thatspecifically bind to MCM2, or to a variant or fragment thereof. Inparticular, the MCM2 antibodies designated as 27C5.6 and 26H6.19 areprovided. Hybridoma cell lines that produce MCM2 monoclonal antibodies27C5.6 and 26H6.19 were deposited with the Patent Depository of theAmerican Type Culture Collection (ATCC), Manassas, Va., 20110-2209 onApr. 14, 2005 and assigned Patent Deposit Nos. PTA-6668 and PTA-6667,respectively. These deposits will be maintained under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. Upon issuance of apatent, Applicants hereby provide assurances that all restrictions onthe accessibility of the above-referenced deposited hybridomas will beirrevocably removed. These deposits were made merely as a conveniencefor those of skill in the art and are not an admission that a deposit isrequired under 35 U.S.C. § 112.

Antibodies that have the binding characteristics of monoclonalantibodies 27C5.6, and 26H6.19 are also disclosed herein. Suchantibodies include, but are not limited to, antibodies that compete incompetitive binding assays with these antibodies, as well as antibodiesthat bind to an epitope capable of binding monoclonal antibody 27C5.6 or26H6.19. Variants and fragments of monoclonal antibodies 27C5.6 and26H6.19 that retain the ability to specifically bind to MCM2 are alsoprovided. Compositions further include hybridoma cell lines that producethe monoclonal antibodies of the present invention and kits comprisingat least one monoclonal antibody disclosed herein.

“Antibodies” and “immunoglobulins” (Igs) are glycoproteins having thesame structural characteristics. While antibodies exhibit bindingspecificity to an antigen, immunoglobulins include both antibodies andother antibody-like molecules that lack antigen specificity.Polypeptides of the latter kind are, for example, produced at low levelsby the lymph system and at increased levels by myelomas.

The terms “antibody” and “antibodies” broadly encompass naturallyoccurring forms of antibodies and recombinant antibodies such assingle-chain antibodies, chimeric and humanized antibodies andmulti-specific antibodies as well as fragments and derivatives of all ofthe foregoing, which fragments and derivatives have at least anantigenic binding site. Antibody derivatives may comprise a protein orchemical moiety conjugated to the antibody. The term “antibody” is usedin the broadest sense and covers fully assembled antibodies, antibodyfragments that can bind antigen (e.g., Fab′, F′(ab)₂, Fv, single chainantibodies, diabodies), and recombinant peptides comprising theforegoing. As used herein, “MCM2 antibody” refers to any antibody thatspecifically binds to MCM2 (SEQ ID NO:1), or to a variant or fragmentthereof, and includes monoclonal antibodies, polyclonal antibodies,single-chain antibodies, and fragments thereof which retain the antigenbinding function of the parent antibody.

The MCM2 antibodies of the invention are optimally monoclonalantibodies. The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (VH) followed by a number of constant domains.Each light chain has a variable domain at one end (V,) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR) regions. The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting ap-sheet configuration, connected by three CDRs, which form loopsconnecting, and 15 in some cases forming part of, the p-sheet structure.The CDRs in each chain are held together in close proximity: by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site: of antibodies (see Kabat et al.,NIH Publ. No. 91-3242, Vol. I, pages 647-669 (1991)).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effecter functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which: are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarily determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institute of Health, i 25Bethesda, Md. [1991]) and/or those residues from a “hypervariable loop”(i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Clothia and Lesk, J. Mol. Biol., 196:901-917[1987]). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein deemed.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (Zapata et al. (1995) ProteinEng. 8(10):1057-1062); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. In a two-chain Fv species, this regionconsists of a dimer of one heavy- and one light-chain variable domain intight, non-covalent association. In a single-chain Fv species, oneheavy- and one light-chain variable domain can be covalently linked byflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy-chain C_(H)1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments that have hinge cysteines betweenthem.

Fragments of the MCM2 antibodies are encompassed by the invention solong as they retain the desired affinity of the full-length antibody.Thus, for example, a fragment of an MCM2 antibody will retain theability to bind to the MCM2 antigen. Such fragments are characterized byproperties similar to the corresponding full-length antibody, that is,the fragments will specifically bind MCM2. Such fragments are referredto herein as “antigen-binding” fragments.

Suitable antigen-binding fragments of an antibody comprise a portion ofa full-length antibody, generally the antigen-binding or variable regionthereof. Examples of antibody fragments include, but are not limited to,Fab, F(ab′)₂, and Fv fragments and single-chain antibody molecules. By“Fab” is intended a monovalent antigen-binding fragment of animmunoglobulin that is composed of the light chain and part of the heavychain. By F(ab′)₂ is intended a bivalent antigen-binding fragment of animmunoglobulin that contains both light chains and part of both heavychains. By “single-chain Fv” or “sFv” antibody fragments is intendedfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. See, forexample, U.S. Pat. Nos. 4,946,778, 5,260,203, 5,455,030, and 5,856,456,herein incorporated by reference. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun (1994) in The Pharmacology of MonoclonalAntibodies, Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, N.Y. ),pp. 269-315.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in, for example,McCafferty et al. (1990) Nature 348:552-554 (1990) and U.S. Pat. No.5,514,548. Clackson et al. (1991) Nature 352:624-628 and Marks et al.(1991) J. Mol. Biol. 222:581-597 describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al. (1992) Bio/Technology10:779-783), as well as combinatorial infection and in vivorecombination as a strategy for constructing very large phage libraries(Waterhouse et al. (1993) Nucleic. Acids Res. 21:2265-2266). Thus, thesetechniques are viable alternatives to traditional monoclonal antibodyhybridoma techniques for isolation of monoclonal antibodies.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al. (1992)Journal of Biochemical and Biophysical Methods 24:107-117 (1992) andBrennan et al. (1985) Science 229:81). However, these fragments can nowbe produced directly by recombinant host cells. For example, theantibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al. (1992) Bio/Technology 10:163-167). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

Preferably antibodies of the invention are monoclonal in nature. Asindicated above, “monoclonal antibody” is intended an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. The term is not limited regarding the species or source of theantibody. The term encompasses whole immunoglobulins as well asfragments such as Fab, F(ab′)2, Fv, and others which retain the antigenbinding function of the antibody. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site, i.e., aparticular epitope within the MCM2 protein, as defined herein below.Furthermore, in contrast to conventional (polyclonal) antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol.222:581-597; and U.S. Pat. No. 5,514,548.

Monoclonal antibodies can be prepared using the method of Kohler et al.(1975) Nature 256:495-496, or a modification thereof. Typically, a mouseis immunized with a solution containing an antigen. Immunization can beperformed by mixing or emulsifying the antigen-containing solution insaline, preferably in an adjuvant such as Freund's complete adjuvant,and injecting the mixture or emulsion parenterally. Any method ofimmunization known in the art may be used to obtain the monoclonalantibodies of the invention. After immunization of the animal, thespleen (and optionally, several large lymph nodes) are removed anddissociated into single cells. The spleen cells may be screened byapplying a cell suspension to a plate or well coated with the antigen ofinterest. The B cells expressing membrane bound immunoglobulin specificfor the antigen (i.e., antibody-producing cells) bind to the plate andare not rinsed away. Resulting B cells, or all dissociated spleen cells,are then induced to fuse with myeloma cells to form monoclonalantibody-producing hybridomas, and are cultured in a selective medium.The resulting cells are plated by serial dilution and are assayed forthe production of antibodies that specifically bind the antigen ofinterest (and that do not bind to unrelated antigens). The selectedmonoclonal antibody (mAb)-secreting hybridomas are then cultured eitherin vitro (e.g., in tissue culture bottles or hollow fiber reactors), orin vivo (as ascites in mice). Monoclonal antibodies can also be producedusing Repetitive Immunizations Multiple Sites technology (RIMMS). See,for example, Kilpatrick et al. (1997) Hybridoma 16(4):381-389; Wring etal. (1999) J. Pharm. Biomed. Anal. 19(5):695-707; and Bynum et al.(1999) Hybridoma 18(5):407-411, all of which are herein incorporated byreference in their entirety.

As an alternative to the use of hybridomas, antibody can be produced ina cell line such as a CHO cell line, as disclosed in U.S. Pat. Nos.5,545,403; 5,545,405; and 5,998,144; incorporated herein by reference.Briefly the cell line is transfected with vectors capable of expressinga light chain and a heavy chain, respectively. By transfecting the twoproteins on separate vectors, chimeric antibodies can be produced.Another advantage is the correct glycosylation of the antibody. Amonoclonal antibody can also be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with a biomarker protein to thereby isolateimmunoglobulin library members that bind the biomarker protein. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP

Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734.

In some aspects of the invention, antibodies may be selected on thebasis of desirable staining of cytological, rather than histological,samples. That is, in particular embodiments the antibodies are selectedwith the end sample type (e.g., cytology preparations) in mind and forbinding specificity. Antibodies directed to specific biomarkers ofinterest, such as MCM2, are selected and purified via a multi-stepscreening process. Such methods for antibody selection are described inpending U.S. application Ser. No. 11/087,227, entitled “Methods andCompositions for the Detection of Cervical Disease,” filed Mar. 23,2005, which is herein incorporated by reference in its entirety.

Antibodies having the binding characteristics of a monoclonal antibodyof the invention are also provided. “Binding characteristics” or“binding specificity” when used in reference to an antibody means thatthe antibody recognizes the same or similar antigenic epitope as acomparison antibody. Examples of such antibodies include, for example,an antibody that competes with a monoclonal antibody of the invention ina competitive binding assay. One of skill in the art could determinewhether an antibody competitively interferes with another antibody usingstandard methods.

By “epitope” is intended the part of an antigenic molecule to which anantibody is produced and to which the antibody will bind. An “MCM2epitope” comprises the part of the MCM2 protein to which an MCM2monoclonal antibody binds. Epitopes can comprise linear amino acidresidues (i.e., residues within the epitope are arranged sequentiallyone after another in a linear fashion), nonlinear amino acid residues(referred to herein as “nonlinear epitopes”; these epitopes are notarranged sequentially), or both linear and nonlinear amino acidresidues. Typically epitopes are short amino acid sequences, e.g. aboutfive amino acids in length. Systematic techniques for identifyingepitopes are known in the art and are described, for example, in U.S.Pat. No. 4,708,871 and in the examples set forth below. Briefly, in onemethod, a set of overlapping oligopeptides derived from the antigen maybe synthesized and bound to a solid phase array of pins, with a uniqueoligopeptide on each pin. The array of pins may comprise a 96-wellmicrotiter plate, permitting one to assay all 96 oligopeptidessimultaneously, e.g., for binding to a biomarker-specific monoclonalantibody. Alternatively, phage display peptide library kits (New EnglandBioLabs) are currently commercially available for epitope mapping. Usingthese methods, the binding affinity for every possible subset ofconsecutive amino acids may be determined in order to identify theepitope that a given antibody binds. Epitopes may also be identified byinference when epitope length peptide sequences are used to immunizeanimals from which antibodies are obtained.

The invention also encompasses isolated polypeptides comprising anepitope for binding an MCM2 monoclonal antibody. These polypeptidescorrespond to a portion of the antigen (i.e., MCM2) that binds to amonoclonal antibody. Such polypeptides find use in methods for producingantibodies that bind selectively to MCM2. The ability of a polypeptideto be used in the production of antibodies is referred to herein as“antigenic activity.” For example, the amino acid sequences set forth inSEQ ID NOs: 3, 4, and 14 (corresponding to residues 369 to 382, 688 to710, and 683 to 692, respectively, in the MCM2 amino acid sequence setforth in SEQ ID NO:1) comprise epitopes recognized by MCM2 monoclonalantibodies, more particularly monoclonal antibodies 27C5.6 and 26H6.19.See Example 4 for details. Variants and fragments of the MCM2 epitopesequences set forth in SEQ ID NOs: 3, 4, and 14 that retain theantigenic activity of the original polypeptide are also provided. Theinvention further includes isolated nucleic acid molecules that encodepolypeptides that comprise MCM2 epitopes, and variants and fragmentsthereof.

The polypeptides of the invention comprising MCM2 epitopes can be usedin methods for producing monoclonal antibodies that specifically bind toMCM2, as described herein above. Such polypeptides can also be used inthe production of polyclonal MCM2 antibodies. For example, polyclonalantibodies can be prepared by immunizing a suitable subject (e.g.,rabbit, goat, mouse, or other mammal) with a polypeptide comprising anMCM2 epitope (i.e., an immunogen). The antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilized biomarkerprotein. At an appropriate time after immunization, e.g., when theantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).

Amino acid sequence variants of a monoclonal antibody or a polypeptidecomprising an MCM2 epitope described herein are also encompassed by thepresent invention. Variants can be prepared by mutations in the clonedDNA sequence encoding the antibody of interest. Methods for mutagenesisand nucleotide sequence alterations are well known in the art. See, forexample, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol.154:367-382; Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and thereferences cited therein; herein incorporated by reference. Guidance asto appropriate amino acid substitutions that do not affect biologicalactivity of the polypeptide of interest may be found in the model ofDayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), herein incorporated byreference. Conservative substitutions, such as exchanging one amino acidwith another having similar properties, may be preferred. Examples ofconservative substitutions include, but are not limited to, Gly

Ala, Val

Ile

Leu, Asp

Glu, Lys

Arg, Asn

Gln, and Phe

Trp

Tyr.

In constructing variants of the polypeptide of interest, modificationsare made such that variants continue to possess the desired activity,i.e., similar binding affinity to the biomarker. Obviously, anymutations made in the DNA encoding the variant polypeptide must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. SeeEP Patent Application Publication No. 75,444.

Preferably, variants of a reference polypeptide have amino acidsequences that have at least 70% or 75% sequence identity, preferably atleast 80% or 85% sequence identity, more preferably at least 90%, 91%,92%, 93%, 94% or 95% sequence identity to the amino acid sequence forthe reference antibody molecule, or to a shorter portion of thereference antibody molecule. More preferably, the molecules share atleast 96%, 97%, 98% or 99% sequence identity. For purposes of thepresent invention, percent sequence identity is determined using theSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrixof 62. The Smith-Waterman homology search algorithm is taught in Smithand Waterman (1981) Adv. Appl. Math. 2:482-489. A variant may, forexample, differ from the reference antibody by as few as 1 to 15 aminoacid residues, as few as 1 to 10 amino acid residues, such as 6-10, asfew as 5, as few as 4, 3, 2, or even 1 amino acid residue.

With respect to optimal alignment of two amino acid sequences, thecontiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. The contiguous segmentused for comparison to the reference amino acid sequence will include atleast 20 contiguous amino acid residues, and may be 30, 40, 50, or moreamino acid residues. Corrections for sequence identity associated withconservative residue substitutions or gaps can be made (seeSmith-Waterman homology search algorithm).

The MCM2 monoclonal antibodies of the invention may be labeled with adetectable substance as described below to facilitate biomarker proteindetection in the sample. Such antibodies find use in practicing themethods of the invention. The antibodies and antibody fragments of theinvention can be coupled to a detectable substance to facilitatedetection of antibody binding. The word “label” when used herein refersto a detectable compound or composition that is conjugated directly orindirectly to the antibody so as to generate a “labeled” antibody. Thelabel may be detectable by itself (e.g., radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition that isdetectable. Examples of detectable substances for purposes of labelingantibodies include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

Kits comprising at least one MCM2 monoclonal antibody of the inventionare further provided. By “kit” is intended any manufacture (e.g., apackage or a container) comprising at least one reagent, i.e., anantibody, for specifically detecting the expression of MCM2. The kit maybe promoted, distributed, or sold as a unit for performing the methodsof the present invention. Additionally, the kits may contain a packageinsert describing the kit and methods for its use.

Kits of the invention generally comprise at least one monoclonalantibody directed to MCM2, chemicals for the detection of antibodybinding, a counterstain, and, optionally, a bluing agent to facilitateidentification of positive staining cells. Any chemicals that detectantigen-antibody binding may be used in the kits of the invention. Insome embodiments, the detection chemicals comprise a labeled polymerconjugated to a secondary antibody. For example, a secondary antibodythat is conjugated to an enzyme that catalyzes the deposition of achromogen at the antigen-antibody binding site may be provided. Suchenzymes and techniques for using them in the detection of antibodybinding are well known in the art. In one embodiment, the kit comprisesa secondary antibody that is conjugated to an HRP-labeled polymer.Chromogens compatible with the conjugated enzyme (e.g., DAB in the caseof an HRP-labeled secondary antibody) and solutions, such as hydrogenperoxide, for blocking non-specific staining may be further provided. Inother embodiments, antibody binding to a biomarker protein is detectedthrough the use of a mouse probe reagent that binds to monoclonalantibodies, followed by addition of a dextran polymer conjugated withHRP that binds to the mouse probe reagent. Such detection reagents arecommercially available from, for example, Biocare Medical.

The kits of the present invention may further comprise a peroxidaseblocking reagent (e.g., hydrogen peroxide), a protein blocking reagent(e.g., purified casein), and a counterstain (e.g., hematoxylin). Abluing agent (e.g., ammonium hydroxide or TBS, pH 7.4, with Tween-20 andsodium azide) may be further provided in the kit to facilitate detectionof positive staining cells. Kits may also comprise positive and negativecontrol samples for quality control purposes.

In another embodiment, the kits of the invention comprise two MCM2monoclonal antibodies, more particularly monoclonal antibodies 27C5.6and 26H6.19. A kit comprising two MCM2 monoclonal antibodies and a thirdantibody directed to topoisomerase II alpha (Topo2A) is furtherprovided. When multiple antibodies are present in the kit, each antibodymay be provided as an individual reagent or, alternatively, as anantibody cocktail comprising all of the antibodies of interest.Furthermore, any or all of the kit reagents may be provided withincontainers that protect them from the external environment, such as insealed containers. The kits of the invention are useful in the diagnosisof high-grade cervical disease and may further include reagents for Papstaining (e.g., EA50 and Orange G).

The compositions of the invention find use in methods for diagnosinghigh-grade cervical disease in a patient such as those disclosed inpending U.S. application Ser. No. 11/087,227, entitled “Methods andCompositions for the Detection of Cervical Disease,” filed Mar. 23,2005, which is herein incorporated by reference in its entirety.“Diagnosing high-grade cervical disease” is intended to include, forexample, diagnosing or detecting the presence of cervical disease,monitoring the progression of the disease, and identifying or detectingcells or samples that are indicative of high-grade cervical disease. Theterms diagnosing, detecting, and identifying high-grade cervical diseaseare used interchangeably herein. By “high-grade cervical disease” isintended those conditions classified by colposcopy as premalignantpathology, malignant pathology, moderate to severe dysplasia, andcervical cancer. Underlying high-grade cervical disease includeshistological identification of CINII, CINIII, HSIL, carcinoma in situ,adenocarcinoma, and cancer (FIGO stages I-IV).

The methods of the invention comprise detecting overexpression of atleast one nuclear biomarker that is selectively overexpressed inhigh-grade cervical disease. By “nuclear biomarker” is intended any geneof protein that is predominantly expressed in the nucleus of the cell. Anuclear biomarker may be expressed to a lesser degree in other parts ofthe cell. By “selectively overexpressed in high-grade cervical disease”is intended that the nuclear biomarker of interest is overexpressed inhigh-grade cervical disease but is not overexpressed in conditionsclassified as LSIL, CINI, HPV-infected samples without any dysplasiapresent, immature metaplastic cells, and other conditions that are notconsidered to be clinical disease. Thus, detection of the nuclearbiomarkers of the invention permits the differentiation of samplesindicative of underlying high-grade cervical disease from samples thatare indicative of benign proliferation, early-stage HPV infection, ormild dysplasia. Nuclear biomarkers of particular interest include MCMproteins, particularly MCM2, and Topo2A.

In a particular aspect of the invention, the methods comprise obtaininga cervical sample from a patient, contacting the sample with at leastone MCM2 monoclonal antibody of the invention, and detecting binding ofthe antibody to MCM2. In other embodiments, the sample is contacted withat least two monoclonal antibodies that specifically bind to MCM2,particularly monoclonal antibodies 27C5.6 and 26H6.19. In a furtherembodiment, the sample is contacted with these two MCM2 monoclonalantibodies and a third antibody that specifically binds to Topo2A.Techniques for detecting antibody binding are well known in the art.Antibody binding to a biomarker of interest may be detected through theuse of chemical reagents that generate a detectable signal thatcorresponds to the level of antibody binding and, accordingly, to thelevel of biomarker protein expression. Any method for detectingantibody-antigen binding may used to practice the methods of theinvention.

As used herein, “cervical sample” refers to any sampling of cells,tissues, or bodily fluids from the cervix in which expression of abiomarker can be detected. Examples of such body samples include but arenot limited to gynecological fluids, biopsies, and smears. Cervicalsamples may be obtained from a patient by a variety of techniquesincluding, for example, by scraping or swabbing an area or by using aneedle to aspirate bodily fluids. Methods for collecting cervicalsamples are well known in the art. In particular embodiments, thecervical sample comprises cervical cells, particularly in a liquid-basedpreparation. In one embodiment, cervical samples are collected accordingto liquid-based cytology specimen preparation guidelines such as, forexample, the SUREPATH® (TriPath Imaging, Inc.) or the THINPREP®preparation (CYTYC, Inc.). Cervical samples may be transferred to aglass slide for viewing under magnification. Fixative and stainingsolutions may be applied to the cells on the glass slide for preservingthe specimen and for facilitating examination. In one embodiment thecervical sample will be collected and processed to provide a monolayersample, as set forth in U.S. Pat. No. 5,346,831, herein incorporated byreference.

One of skill in the art will appreciate that any or all of the steps inthe methods of the invention could be implemented by personnel in amanual or automated fashion. Thus, the steps of cervical samplepreparation, antibody, and detection of antibody binding may beautomated. The methods of the invention may also be combined withconventional Pap staining techniques to permit a more accurate diagnosisof high-grade cervical disease.

The following examples are offered by way of illustration and not by wayof limitation:

EXPERIMENTAL Example 1 Production of Mouse Monoclonal Antibodies to MCM2

Mouse monoclonal antibodies specific for MCM2 were generated. Theantigen (an immunogenic polypeptide) was a full-length recombinanthexahistidine-tagged MCM2 protein. The antigen was expressed using abaculovirus expression system in Tni cells. Specifically, the codingsequence for the hexahistidine-tagged MCM2 (SEQ ID NO:10) was clonedinto the pFastBac1 plasmid (Invitrogen) for expression in Tni cells.Methods for producing recombinant proteins using baculovirus expressionsystems are well known in the art. The tagged MCM2 protein was purifiedusing a chelating agarose charged with Ni+2 ions (Ni-NTA from Qiagen)and used as an immunogen. The amino acid sequence of the immunogenicMCM2 polypeptide is provided in SEQ ID NO:11.

Mouse immunizations and hybridoma fusions were performed essentially asdescribed in Kohler et al. (1975) Nature 256:495-496. Mice wereimmunized with the immunogenic tagged-MCM2 protein in solution.Antibody-producing cells were isolated from the immunized mice and fusedwith myeloma cells to form monoclonal antibody-producing hybridomas. Thehybridomas were cultured in a selective medium. The resulting cells wereplated by serial dilution and assayed for the production of antibodiesthat specifically bind MCM2 (and that do not bind to unrelatedantigens). To confirm that the monoclonal antibodies of interest reactedwith the MCM2 protein only and not with the hexahistidine tag, selectedhybridomas were screened against an MCM2-FLAG-tagged protein. Thenucleotide and amino acid sequences for the MCM2-FLAG protein are setforth in SEQ ID NOs:12 and 13, respectively. Selected monoclonalantibody (mAb)-secreting hybridomas were then cultured.

Antibodies were purified from the culture media supernatants of“exhausted” hybridoma cells (i.e., cells grown until viability drops tobetween 0-15%) using recombinant Protein A-coated resin (STREAMLINE®,Amersham, Inc.). Antibodies were eluted using low pH followed byimmediate neutralization of pH. Fractions with significant absorbancesat 280 nM were pooled. The resultant pool was dialyzed against PBS.Purified antibodies were subjected to further characterization. MCM2monoclonal antibodies 26H6.19 and 27C5.6 were both determined to be IgG₁isotypes. Details of the epitope mapping of these antibodies aredescribed below.

Example 2 Isolation of Monoclonal Antibodies from Hybridoma Cells

The following procedure is used to isolate monoclonal antibodies fromhybridoma cells:

Media Preparation

-   -   To a sterile 1,000 ml storage bottle, add 100 ml Hyclone Fetal        Bovine Serum (FBS).    -   Add 10 ml of MEM Non-Essential Amino Acids Solution.    -   Add 10 ml of Penicillin-Streptomycin-L-Glutamine Solution.    -   QS to approximately 1000 ml with ExCell 610-HSF media.    -   Place sterile cap on bottle and secure tightly. Swirl gently to        mix.    -   Connect a 1000 ml sterile acetate vacuum filter unit (0.2 μm) to        a vacuum pump system.    -   Gently pour approximately half of the media solution into        sterile acetate vacuum filter unit and turn on the vacuum.    -   Once the first half of the media has been filtered, pour the        remaining media into the filter unit and continue filtering.    -   After all the media has been filtered, disconnect the vacuum        hose from the vacuum filter unit and turn off the vacuum pump.        Remove the receiver portion of the filter unit from the filter        bottle. Place a new sterile bottle cap on the bottle.    -   Store at 2° C. to 10° C. Protect from light.        Initial Hybridoma Cell Culture    -   Thaw vial of stock hybridoma frozen culture in a pre-warmed        37° C. H₂O bath.    -   Spray the outside of the freeze vial with 70% ethanol.    -   Move the thawed vial into the Biological Safety Cabinet.    -   Remove the cells from the freeze vial and transfer the cells to        a 15 ml centrifuge tube.    -   Add 7 ml of cell culture media drop-wise to the 15 ml centrifuge        tube containing the thawed cells.    -   Centrifuge the 15 ml centrifuge tube containing the thawed cells        and culture media for 5 minutes at 200 g force.    -   While the cells are in the centrifuge, add 45 ml of cell culture        media to a sterile T-225 flask.    -   After centrifugation, visually inspect the tube for the presence        of a cell pellet.    -   Remove the media from the centrifuge tube being careful not to        dislodge the cell pellet. Note: If the cell pellet is disturbed,        repeat the centrifugation step.    -   Add 5 ml of cell culture media to the 15 ml centrifuge tube        containing the pelleted cells. Pipette to re-suspend the cell        pellet into the media.    -   Transfer the entire contents of the resuspended cells and        culture media into the T-225 flask containing the 45 ml of        media.    -   Cap the T-225 flask.    -   Observe for presence of intact cells under the microscope. Place        the T-225 flask immediately into a CO2 incubator and allow the        cells to incubate overnight.        Expansion of Hybridoma Cell Line    -   Continue to monitor the cell culture for viability,        concentration, and presence of contamination.    -   Monitor and adjust the cell suspension from the initial T-225        flask until the concentration is approximately 600,000 cells/ml        to 800,000 cells/ml and a total of 200 to 250 ml of media.    -   Dislodge cells and add additional media as needed to meet        minimum cell density requirements. Divide and transfer cell        suspension into one new sterile T-225 flask. Place the 2×T-225        flasks into the CO2 incubator.    -   Monitor the cells from the 2×T-225 flasks until the        concentration is approximately 600,000 cells/ml to 800,000        cells/ml, and a total of between 200 to 250 ml of media for each        flask.    -   Dislodge cells and add additional media as needed to meet        minimum cell density requirements. Divide and transfer the cell        suspensions into 2 additional new sterile T-225 flasks for a        total of 4×T-225 flasks. Return all flasks to the CO2 incubator.    -   Monitor the cells, and adjust volume in the 4×T-225 flasks until        the cell concentration is approximately 600,000 cells/ml to        800,000 cells/ml with a total volume of approximately 250 ml per        T-225 flask (or approximately 1000 ml total).    -   Continue to monitor the cells from the 4×T-225 flasks until the        cells have grown to exhaustion, with a final viability of        0%-15%. The cell culture supernatant is now ready for the        Clarification Process.        Clarification of Supernatant    -   Turn on the tabletop centrifuge. Place the 500 ml tube adapters        into the rotor buckets, close the lid and set the temperature to        4° C. (+/−) 4° C.    -   Using aseptic technique, pour the media from all four of the now        exhausted T-225 flasks into 2×500 ml conical centrifuge tubes.    -   Make sure the 2×500 ml tubes are balanced. Transfer supernatant        from one tube to the other as necessary to balance them.    -   Centrifuge the exhausted supernatant at 1350 g (+/−40 g) for 15        minutes at 2° C. to 10° C.    -   After centrifugation is complete, aseptically decant the        supernatant into a sterile 1000 ml storage bottle and secure        with a sterile cap.    -   Aseptically transfer 1 ml to the microfuge tube. Store microfuge        tube with sample at 2° C. to 10° C. (Protect from light).    -   The clarified supernatant sample is ready for IgG evaluation        using the Easy-Titer® Assay.        Buffer Preparation        Binding Buffer:    -   Add approximately 600 ml of DI H₂O to a clean beaker.    -   Add 77.28 ml of Boric Acid solution (4% W/V). Stir at room        temperature with a clean stir bar.    -   Weigh out 233.76 g of Sodium Chloride and place into the        solution while continuing to stir.    -   Bring solution up to approximately 950 ml with DI H₂O and        continue to stir.    -   When the Sodium Chloride has dissolved and the solution is        clear, adjust the pH to 9.0±0.2 with Sodium Hydroxide.    -   Remove the solution to a clean 1000 ml graduated cylinder and QS        to 1000 ml with DI H₂O.    -   Transfer the completed buffer to an appropriate storage bottle.        This buffer may be stored for up to 7 days before use.    -   Repeat this entire process to prepare an additional 0.2 liters        to 1.0 liter of Binding Buffer.        Elution Buffer    -   Weigh out 1.725 g of sodium phosphate, monobasic and place into        a clean 250 ml beaker with a clean stir bar.    -   Weigh out 3.676 g of sodium citrate and place into the same        clean 250 ml beaker.    -   Add approximately 175 ml of DI H₂O and stir at room temperature        until dissolved.    -   Weigh out 4.38 g of Sodium Chloride and place into the solution        while continuing to stir.    -   Bring solution up to approximately 225 ml with DI H₂O and        continue to stir.    -   When the Sodium Chloride has dissolved and the solution is        clear, adjust the pH to 3.5±0.2 with Hydrochloric Acid.    -   Remove the solution to a clean 250 ml graduated cylinder and QS        to 250 ml with DI H₂O.    -   Connect a 500 ml sterile acetate vacuum filter unit (0.2 μm) to        a vacuum pump system and filter sterilize the solution.    -   Remove the filter and close the container with a sterile cap.        Antibody Adsorption    -   Pour the Clarified Supernatant (˜1 L) into a clean 4000 ml        plastic beaker with a clean stir bar.    -   Add an approximately equal amount (˜1 L) of the Binding Buffer        to the clean 4000 ml plastic beaker containing the clarified        supernatant. Add a clean stir bar.    -   Cover the beaker with clean plastic wrap and label “Antibody        Binding.”    -   Calculate the approximate amount of STREAMLINE® Protein A that        will be needed using the data in Table 1.

TABLE 1 Volume of Protein A Resin Required Volume of Protein A QuantityIgG (μg/ml) Resin Required in in Supernatant Milliliters (ml) >180-≦20012.0 >160-≦180 11.0 >140-≦160 10.0 >120-≦140 9.0 >100-≦120 8.0  >80-≦1007.0 >60-≦80 6.0 >40-≦60 4.5 >20-≦40 3.5 ≦20 2.0

-   -   Secure a clean Disposable Column and stopcock assembly to a ring        stand and clamp. Close the stopcock.    -   Mix appropriate amount of STREAMLINE® Protein A beads by        inverting the bottle several times. Withdraw the required volume        and place into the Disposable Column.    -   Wash the STREAMLINE® Protein A beads with 10 ml of DI H₂O. Open        the stopcock and allow the DI H₂O to drain. Close the stopcock.        Repeat with an additional 10 ml of DI H₂O.    -   Wash the STREAMLINE® Protein A beads with 10 ml of Binding        Buffer. Open the stopcock and allow the Binding Buffer to drain.        Close the stopcock. Repeat with an additional 10 ml of Binding        Buffer.    -   Resuspend the STREAMLINE® Protein A beads in ˜10 ml of the        Clarified Supernatant and Binding Buffer solution (from the 4000        ml beaker) and transfer the beads into the 4000 ml beaker        containing the Clarified Supernatant and Binding Buffer        solution. Repeat as required to transfer any remaining beads.        When completed, discard the column and stopcock.    -   Allow the mixture to mix vigorously at 2° C. to 10° C. for        approximately 18 hours.    -   When mixing is complete, turn off the stir plate and remove the        “Antibody Binding” beaker with the buffered supernatant and bead        suspension back to the lab bench area. Allow the STREAMLINE®        Protein A beads to settle to the bottom of the beaker        (approximately 5 minutes).    -   Secure a clean Disposable Column and stopcock assembly to a ring        stand and clamp. Close the stopcock.    -   Label a clean, 250 ml bottle or suitable container “Column        Wash-Post Binding.”    -   Label a clean plastic beaker “Supernatant-Post Binding.”    -   Decant the supernatant from the 4000 ml beaker into the clean,        labeled, 2 liter plastic beaker, leaving the beads in the bottom        of the 4000 ml beaker. Cover the 2000 ml beaker containing the        “Supernatant-Post Binding” solution with clean plastic wrap and        store at 2° C. to 10° C.    -   Add approximately 15 ml of Binding Buffer into the decanted 4000        ml “Antibody Binding” beaker. Resuspend the STREAMLINE® Protein        A beads and transfer them to the column. Open the stopcock and        allow the Binding Buffer to drain into the “Column Wash-Post        binding” container. Close the stopcock when drained.    -   Transfer any remaining STREAMLINE® Protein A beads in the        “Antibody Binding” beaker by adding additional Binding Buffer,        mixing, and transferring to the column as in the preceding        steps. Close the stopcock when drained.    -   Calculate the approximate amount of Binding Buffer needed to        wash the STREAMLINE® Protein A beads in the column using the        data in Table 2.

TABLE 2 Binding Buffer Volume for Column Wash Volume of Binding QuantityIgG (μg/ml) Buffer Required in in Supernatant Milliliters (ml) >180-≦2005 column washes total with 15.0 ml each >160-≦180 5 column washes totalwith 15.0 ml each >140-≦160 5 column washes total with 12.5 mleach >120-≦140 5 column washes total with 12.5 ml each >100-≦120 5column washes total with 12.5 ml each  >80-≦100 5 column washes totalwith 10.0 ml each >60-≦80 5 column washes total with 10.0 mleach >40-≦60 5 column washes total with 7.5 ml each >20-≦40 5 columnwashes total with 5.0 ml each ≦20 5 column washes total with 5.0 ml each

-   -   Wash the STREAMLINE® Protein A beads in the column with the        appropriate volume of Binding Buffer for the appropriate number        of washes, continuing to collect the efluent into the “Column        Wash-Post Binding” container.    -   When completed, close the stopcock. Store the “Column Wash-Post        Binding” container at 2° C. to 10° C.    -   Determine the Total Volumes of Elution Buffer and Neutralization        Buffer needed to elute the STREAMLINE® Protein A beads in the        column from Table 3.

TABLE 3 Determination of Amount of Elution Buffer and NeutralizationBuffer Volume of Volume of Quantity IgG Total Volume of Total Volume ofElution Buffer Neutralization (μg/ml) in Elution Buffer NeutralizationRequired per Buffer Required per Supernatant Required (ml) BufferRequired (ml) fraction (ml) fraction (ml) >180-≦200 72 7.2 121.2 >160-≦180 66 6.6 11 1.1 >140-≦160 60 6.0 10 1.0 >120-≦140 54 5.4 90.9 >100-≦120 48 4.8 8 0.8  >80-≦100 42 4.2 7 0.7 >60-≦80 36 3.6 60.6 >40-≦60 27 2.7 4.5 0.45 >20-≦40 21 2.1 3.5 0.35 ≦20 12 1.2 2 0.2

-   -   Label 9 sterile conical centrifuge tubes “Eluted Antibody”,        Fraction # (1 through 9).    -   Place the appropriate volume of Neutralization Buffer required        per fraction (as determined from Table “C” above) into each of        the 9 “Eluted Antibody” fraction tubes and place securely under        the column stopcock outlet.    -   Elute the STREAMLINE® Protein A beads in the column fraction by        fraction with the appropriate volume of Elution Buffer required        per fraction (as determined from Table 3 above) while collecting        the eluate into each of the “Eluted Antibody” tubes containing        Neutralization Buffer.    -   When the elutions are complete, mix each “Eluted Antibody”        fraction tube gently by swirling several times. Remove        approximately 50 μl of fraction # 3 and place on a pH test paper        strip to ensure that the eluate has been neutralized to an        approximate pH between 6.5 to 8.5. If required, add additional        Neutralizing Buffer or Elution Buffer as needed to bring pH into        range.    -   When pH evaluation is completed, perform an Absorbance Scan of a        sample from each fraction at 280 nm-400 nm to determine the        approximate concentration of IgG in the eluate prior to        proceeding to the Dialysis Process.        -   Accept fractions as part of the Eluate Pool if the A280-A400            value is ≧0.200.        -   Reject fractions as part of the Eluate Pool if the A280-A400            value is <0.200.    -   Label a sterile conical centrifuge tube “Eluted Antibody,”        “Eluate Pool,” and combine all fractions that were Accepted as        part of the pool.    -   Perform an Absorbance Scan of a sample of the Eluate Pool to        determine the approximate concentration of IgG in the eluate        prior to proceeding to the Dialysis Process.    -   Estimate the volume of the Eluate Pool and calculate the        approximate total mgs of IgG.    -   Volume of Eluate Pool:______mls×______IgG mg/ml=______Total mgs        of IgG        Antibody Dialysis    -   Remove the “Eluted Antibody” tube from 2° C. to 10° C.    -   Calculate the approximate length of Dialysis Tubing that will be        needed to dialyze the antibody eluate using the approximate        volume of eluate and the data in Table 4.

TABLE 4 Calculation of Length of Dialysis Tubing Needed ApproximateLength Approximate Approximate Needed for Length Approximate ApproximateVolume/length Length Needed Head Space Sample plus Needed for TotalLength of Volume of Ratio of Dialysis for Eluent of 20% Headspace TieOff of Dialysis Tubing Eluent (ml) Tubing Sample (cm) (cm) (cm) Tubing(cm) Needed (cm) 39.6 2 20 4 24 15 63 36.3 2 18 4 22 15 59 33.0 2 17 320 15 55 29.7 2 15 3 18 15 51 26.4 2 13 3 16 15 47 23.1 2 12 2 14 15 4319.8 2 10 2 12 15 39 14.85 2 7 1 9 15 33 11.55 2 6 1 7 15 29 6.6 2 3 1 415 23

-   -   Cut the appropriate length of dialysis tubing required.        (Spectra/Por® 2 Regenerated Cellulose Membrane, 12,000-14,000        Dalton Molecular Weight Cutoff (MWCO), 16 mm Diameter, Spectrum        Laboratories Inc., Cat. No. 132678)    -   Hydrate the dialysis membrane tubing in 1000 ml of DIH₂O for >30        minutes.    -   Calculate the approximate volume of Dialysis Buffer needed to        dialyze the antibody eluate using the data in Table 5.

TABLE 5 Volume of Dialysis Buffer Required Quantity IgG Final Volume ofLength of Volume of Dialysis (μg/ml) in Eluted Antibody Dialysis TubingBuffer (1 × PBS) Supernatant in Milliliters (ml) Needed (cm) Needed inLiters >180-≦200 39.6 ml 63 cm 3 complete changes of 4.0Liters >160-≦180 36.3 ml 59 cm 3 complete changes of 3.6Liters >140-≦160 33.0 ml 55 cm 3 complete changes of 3.3Liters >120-≦140 29.7 ml 51 cm 3 complete changes of 3.0Liters >100-≦120 26.4 ml 47 cm 3 complete changes of 2.6 Liters >80-≦100 23.1 ml 43 cm 3 complete changes of 2.3 Liters >60-≦80 19.8 ml39 cm 3 complete changes of 1.9 Liters >40-≦60 14.85 ml  33 cm 3complete changes of 1.5 Liters >20-≦40 11.55 ml  29 cm 3 completechanges of 1.2 Liters ≦20  6.6 ml 23 cm 3 complete changes of 0.7 Liters

-   -   Place the appropriate amount of Dialysis Buffer into a suitable        sized plastic beaker. Label the beaker “Dialyzed Antibody.” Add        a clean stir bar and place the beaker on a stir plate inside a        refrigerator or cold room at 2° C. to 10° C.    -   Rinse the dialysis tubing thoroughly in DI-H₂O. Tie two end        knots approximately 7 cm from one end of the dialysis tubing and        secure tightly.    -   Add approximately 5 ml of DI-H₂O into the dialysis tubing.    -   Fill the dialysis tubing with the eluted antibody from the        “Eluted Antibody” collection tube.    -   Tie two end knots approximately 7 cm from the remaining open end        of the dialysis tubing and secure tightly. Ensure that the        headspace is approximately that as derived from Table 4.    -   Place the filled and closed dialysis tubing into the dialysis        reservoir with the appropriate volume of 1×PBS (from Table 5).    -   Cover the beaker with clean plastic wrap. Adjust the speed on        the stir plate such that the dialysis sample spins freely, but        is not pulled down into the vortex of the dialysate. Dialysis        should take place at 2° C. to 10° C. with 3 buffer exchanges in        total within a 24 hour period.        Antibody Filtration    -   Label a sterile collection tube “Dialyzed Antibody.”    -   Remove the dialyzed sample tubing from the dialysis beaker. Cut        the dialysis tubing open at one end and transfer the dialyzed        sample into the “Dialyzed Antibody” centrifuge tube.    -   Label another sterile collection tube “Dialyzed Antibody.”    -   Select a sterile Luer Lok syringe with adequate capacity to hold        the final dialyszed volume.    -   Attach an ACRODISC® Syringe Filter to the opening of the syringe        (0.2 μm HT TUFFRYN® Membrane, Low Protein binding, Gelman        Laboratories, Cat. No. 4192). Remove the plunger from the        syringe and while holding the syringe upright, transfer the        dialyszed monoclonal antibody from the “Dialyzed Antibody” tube        into the syringe. Replace the plunger.    -   Hold the ACRODISC® Syringe Filter over the opened, sterile,        labeled “Purified Antibody” collection tube, and depress the        syringe plunger to filter the purified antibody into the        “Purified Antibody” tube.    -   When filtration is complete, cap the “Purified Antibody” tube        and store at 2° C. to 10° C.    -   Determine concentration of purified monoclonal antibody using        A280 procedure.

Example 3 General Method for Epitope Mapping

General Approach

Epitope mapping is performed to identify the linear amino acid sequencewithin an antigenic protein (i.e., the epitope) that is recognized by aparticular monoclonal antibody. A general approach for epitope mappingrequires the expression of the full-length protein, as well as variousfragments (i.e., truncated forms) of the protein, generally in aheterologous expression system. These various recombinant proteins arethen used to determine if the specific monoclonal antibody is capable ofbinding one or more of the truncated forms of the target protein.Through the use of reiterative truncation and the generation ofrecombinant proteins with overlapping amino acid regions, it is possibleto identify the region that is recognized by the monoclonal antibodyunder investigation. Western blot analysis or ELISA is employed todetermine if the specific monoclonal antibody under investigation iscapable of binding one or more of the recombinant protein fragments.This approach can ultimately identify the peptide regions that containsthe epitope and, in some cases, to refine the epitope precisely to an8-11 amino acid sequence.

Construct Design and Creation

The first step in epitope mapping is the design of nested genetruncations. Frequently, the gene is divided into four equal parts forfurther analysis.

Gene Cloning Strategy

The general cloning strategy begins with PCR-based generation of thecloned gene fragments. In order to efficiently express the clonedfragment, especially when using small amino acid regions, the clonedfragment is expressed as a fusion protein, i.e. fused to another carrierprotein that is stably expressed in the system. Green fluorescentprotein (GFP) is frequently used as the carrier protein. GFP is includedas a fusion partner to stabilize the truncation fragments and improveexpression during the subsequent in vitro protein expression step. GFPalso permits the tracking of fusion-protein expression using anti-GFPantibodies.

Cloning to create the GFP-protein construct is performed using eitherthe mega-priming approach or through the use of plasmid cloning into thepScreen-GFP vector. Generally, the truncation fragments are fused to GFPand control sequences necessary for protein expression using a techniquecalled megapriming.

Megapriming is the joining of two or more DNA fragments by annealinghomologous regions at the end of the respective fragments and extendingthe annealed single-stranded DNA with a thermostable DNA polymerase.This process creates one large DNA fragment from two or more smallerfragments, linking them by their shared sequence. This large fragment isthen amplified using standard PCR.

If megapriming cannot be used successfully, the truncation fragments canbe cloned into a plasmid containing GFP and protein-expression controlsequences. This cloning creates the GFP/fragment fusions necessary forepitope mapping. The remainder of the protocol can then proceed asdescribed below.

Protein Expression

The expression constructs created by, for example, megapriming are thenintroduced into the Rapid Translation System (RTS). RTS is a cell-freeprotein expression system derived from E. coli lysates. This systempermits rapid (3-4 hour) expression of proteins from DNA templates.

If RTS does not produce adequate levels of protein expression, then thetruncation fragments will be cloned into the GFP protein-expressionplasmid. These fusion plasmids are then transformed into an E. colistrain optimized for protein expression. Protein expression is inducedin a growing culture of bacteria and, following outgrowth, the cells arelysed. The proteins in the complex cell lysate are then separated bypolyacrylamide gel electrophoresis (PAGE), and the remainder of theprotocol is the same as below.

Protein Detection and Epitope Mapping

Protein fragments produced by RTS are separated using PAGE andtransferred onto nitrocellulose membranes. The membrane-bound proteinsare then exposed to the antibody under investigation in solution.Antibody/protein binding is identified using colorimetric techniquesknown in the art.

Antibody binding of the full-length protein and some subset of thetruncated protein fragments constitutes a positive result. If theabsence of a particular section of the protein eliminates antibodybinding, then the epitope lies on this fragment.

If the antibody to be mapped does not recognize protein bound tonitrocellulose membranes, then alternative methods for detectingantibody/protein interactions, such as, for example, ELISA orimmunoprecipitation are used. Methods for detecting antibody/proteininteractions are well known in the art.

Refining the Epitope Location

Since the above-described protocol will only narrow the location of theepitope down to approximately one-quarter of the protein, it isnecessary to repeat the process on the quarter of the protein determinedto contain the epitope in order to further resolve the location of theepitope. For a very large protein, it may be necessary to repeat thisprocess two to three times to narrow the epitope down to 8-15 aminoacids.

Example 4 Characterization of Epitopes for MCM2 Monoclonal Antibodies27C5.6 and 26H6.19

Epitope mapping for MCM2 Monoclonal Antibodies 27C5.6 and 26H6.19 wascarried out essentially as described in Example 3. Specifically, PCR wasused to create MCM2 gene truncations, followed by RTS to generaterecombinant MCM2 protein fragments, and finally western blotting todetect antibody binding to MCM2. GFP was joined with the MCM2 genetruncations in a second round of PCR to ensure robust and stableexpression in RTS.

The full-length coding sequence for MCM2 (SEQ ID NO:2; NM_(—)004526) hasa size of 2715 bp. However, the cDNA that was used to express therecombinant MCM2 protein and that was used to immunize mice during theproduction of MCM2 antibodies had a gene size of 2688 bp (SEQ ID NO:5).The truncated MCM2 cDNA used had a 27 bp region missing at the 5′ end ofthe MCM2 protein, specifically the fragment ATGGCGGAATCATCGGAATCCTTCACC(SEQ ID NO:6). The following sequential steps were carried out in orderto epitope map the MCM2-27C5.6 antibody:

Since the MCM2 gene was large (>1000 bp) and to minimize the number ofiterations of PCR needed, the gene was equally divided into six regions[1-6] of approximately 400 bp. Overlapping sequences, which containhomologous sequence to permit mega priming during a second PCR cycle andrestriction sites for a second option of sub-cloning into pScreen-GFPplasmid, were added to the gene of interest during the first PCR. Thefirst round of PCR created fragments of the truncated MCM2 nucleotidesequence (SEQ ID NO:5) including: region [1] was 1-426 bp, region [1-2]was 1-888 bp, region [1-3] was 1-1377 bp, region [1-4] was 1-1845 bp,region [1-5] was 1-2241 bp, region [1-6] was 1-2688 bp, and finallyregion [2-6] was 427-2688 bp. Individual regions (example region [5])were not expressed to avoid missing epitopes that were present injunction sequence between regions.

The first round PCR products of MCM2 were subcloned into pSCREEN-GFP(BamH1-Xho1), as the fragment sizes were too large for mega-priming. Theonly truncation that was unsuccessful was the full length region [1-6].The original primers used to amplify the full-length gene andtruncations were engineered to include restriction sites (5′ end BAMH1;3′end XHO1) to allow direct subcloning into pSCREEN-GFP.

The GFP-gene fusions created were used as a template for proteinproduction in the RTS reaction using the RTS 100 E. coli HY kit fromRoche. The protein products from RTS were acetone precipitated, loadeddirectly onto a denaturing polyacrylimide gel, and analyzed by westernblotting. The western blot was probed directly with the 27C5.6monoclonal antibody and GFP antibodies.

The first round of RTS products were probed with both GFP antibodies andthe MCM2 monoclonal antibody 27C5.6. A positive band was detected inregion [1-3]. The above process was repeated using the fragmentencompassed by region [1-3] as the starting sequence.

A second round of RTS produced a positive result for the 27C5.6 antibodyin the region MCM2-3Q3 (CQSAGPFEVNMEETIYQNYQRIRIQESP (SEQ ID NO:7);corresponding to amino acid residues 355 to 382 of SEQ ID NO:1). Theabove process was repeated using the fragment encompassed by regionMCM2-3Q3 as the starting sequence.

A third round of RTS produced a positive result for the 27C5.6 antibodyin the region MCM2-3Q3.2 (IYQNYQRIRIQESP (SEQ ID NO:3); corresponding toamino acid residues 369 to 382 of SEQ ID NO:1). No positive result wasobtained in region MCM2-3Q3.1 (CQSAGPFEVNMEET (SEQ ID NO:8);corresponding to amino acid residues 355 to 368 of SEQ ID NO:1) or inMCM2-3Q3.2 (EVNMEETIYQNYQR (SEQ ID NO:9); corresponding to amino acidresidues 362 to 375 of SEQ ID NO:1).

Results

Initial results showed that the epitope for the MCM2 monoclonal antibody27C5.6 is located within the N-terminal region of the MCM2 protein.Continued truncations of the MCM2 protein showed that the epitoperecognized by 27C5.6 is located within a fourteen amino acid region,specifically corresponding to amino acid residues 369-382 of SEQ ID NO:1(IYQNYQRIRIQESP (SEQ ID NO:3)). Additional rounds of RTS may be able torefine the epitope location further.

The identical process described above was used to identify the epitopefor MCM2 monoclonal antibody 26H6.19. Initial results indicated that theepitope was located within the C-terminal region of the MCM2 protein.The epitope was preliminarily defined to a twenty-three amino acidregion, specifically corresponding to amino acid residues 688-710 of SEQID NO:1 (PSNKEEEGLANGSAAEPAMPNTY (SEQ ID NO:4)). Further analysisrefined the epitope of MCM2 monoclonal antibody 26H6.19 to a ten aminoacid region comprising amino acid residues 683-692 of SEQ ID NO:1(HVRHHPSNKE (SEQ ID NO:14)).

1. A monoclonal antibody that is capable of specifically binding toMCM2, wherein the antibody is selected from the group consisting of: (a)the monoclonal antibody produced by the hybridoma cell line 27C5.6,deposited with the ATCC as Patent Deposit No. PTA-6668; (b) themonoclonal antibody produced by the hybridoma cell line 26H6.19,deposited with the ATCC as Patent Deposit No. PTA-6667; (c) a monoclonalantibody that binds to the amino acid sequence set forth in SEQ ID NO:3;(d) a monoclonal antibody that binds to the amino acid sequence of SEQID NO:14; and (e) an antigen binding fragment of a monoclonal antibodyof (a)-(d), wherein the fragment retains the capability of specificallybinding to MCM2.
 2. The hybridoma cell line 27C5.6, deposited with theATCC as Patent Deposit No. PTA-6668.
 3. The hybridoma cell line 26H6.19,deposited with the ATCC as Patent Deposit No. PTA-6667.
 4. A hybridomacell line capable of producing a monoclonal antibody of claim
 1. 5. Akit for diagnosing high-grade cervical disease comprising at least onemonoclonal antibody according to claim
 1. 6. The kit of claim 5, whereinthe monoclonal antibody is the monoclonal antibody produced by thehybridoma cell line 27C5.6, deposited with the ATCC as Patent DepositNo. PTA-6668, or the monoclonal antibody produced by the hybridoma cellline 26H6.19, deposited with the ATCC as Patent Deposit No. PTA-6667. 7.The kit of claim 5 comprising at least two antibodies, wherein a firstantibody is the monoclonal antibody produced by the hybridoma cell line27C5.6, deposited with the ATCC as Patent Deposit No. PTA-6668, and asecond antibody is the monoclonal antibody produced by the hybridomacell line 26H6.19, deposited with the ATCC as Patent Deposit No.PTA-6667.
 8. The kit of claim 7 further comprising an antibody thatspecifically binds to Topo2A.
 9. The kit of claim 7, wherein eachantibody is provided as a separate antibody reagent.
 10. The kit ofclaim 7, wherein all of the antibodies are provided as an antibodycocktail.
 11. The kit of claim 5 further comprising reagents for Papstaining.
 12. The kit of claim 11, wherein the reagents for Pap stainingcomprise EA50 and Orange G.
 13. The monoclonal antibody of claim 1,wherein the monoclonal antibody is the monoclonal antibody produced bythe hybridoma cell line 27C5.6, deposited with the ATCC as PatentDeposit No. PTA-6668.
 14. The monoclonal antibody of claim 1, whereinthe monoclonal antibody is the monoclonal antibody produced by thehybridoma cell line 27C5.6, deposited with the ATCC as Patent DepositNo. PTA-6667.